The nature of the human T cell response to the cancer antigen 5T4 is determined by the balance of regulatory and inflammatory T cells of the same antigen-specificity: implications for vaccine design

Abstract

The oncofoetal antigen 5T4 is a promising T cell target in the context of colorectal cancer, as demonstrated by a recent clinical study where 5T4-specific T cell responses, induced by vaccination or cyclophosphamide, were associated with a significantly prolonged survival of patients with metastatic disease. Whilst Th1-type (IFN-γ⁺) responses specific to 5T4, and other oncofoetal antigens, are often readily detectable in early stage CRC patients and healthy donors, their activity is suppressed as the cancer progresses by CD4⁺CD25^hiFoxp3⁺ regulatory T cells (Treg) which contribute to the immunosuppressive environment conducive to tumour growth. This study mapped the fine specificity of Th1 and Treg cell responses to the 5T4 protein. Surprisingly, both immunogenic peptides and those recognised by Tregs clustered in the same HLA-DR transcending epitope-rich hotspots within the 5T4 protein. Similarly, regions of low Th1-cell immunogenicity also did not contain peptides capable of stimulating Tregs, further supporting the notion that Treg and Th1 cells recognise the same peptides. Understanding the rules which govern the balance of Th1 and Treg cells responding to a given peptide specificity is, therefore, of fundamental importance to designing strategies for manipulating the balance in favour of Th1 cells, and thus the most effective anti-cancer T cell responses.

Keywords: 5T4; Antigen specificity; Cancer; Regulatory T cells; T cells.

Fig. 1

Mapping HLA-DR-restricted 5T4 epitopes. a Forty-one 20mer peptides, overlapping by 10 amino acids and spanning the entire 5T4 protein, were incorporated into a peptide pool matrix, whereby each peptide was placed in two pools to aid identification. b Following short-term culture of PBMC isolated from healthy donors (n = 9) and CRC patients (n = 16), 5T4-specific T cell responses were measured by IFN-γ ELISpot, and the number of IFN-γ⁺ spot-forming cells (SFC) per 10⁵ cultured PBMC to each peptide pool collated. c HLA-DR heatmap analysis was performed on all patients and healthy donors shown in b, in addition to patients and healthy donors used in an earlier study where identical peptide pool experiments were performed [15]. Grey boxes indicate where a putative 5T4-specific response (positive according to pooling matrix analysis) has been identified in that individual, example is shown for all HLA-DRB1*01⁺ donors. Donor status is indicated by the coloured box, green (healthy donors) and orange (cancer patients). d This analysis was repeated for all individuals stratified by HLA-DRB1-type. The heatmap illustrates the proportion of individuals putatively responding to each 5T4 peptide

Fig. 2

Characterising peptide-specific 5T4 T cell responses generated by healthy donors and CRC patients. The percentage of healthy donors and CRC patients who had positive responses to each candidate 5T4 peptide (a) and the response rate to all peptides (b). c The mean IFN-γ⁺ T cell response to each candidate peptide, defined as the number of SFC/10⁵ cultured PBMC, amongst healthy donors and CRC patients. The mean T cell response and response rate (%) amongst healthy donors (d) and CRC patients (e) is graphed together. Peptides which are recognised with high frequency (> 50%) in healthy donors are highlighted in green. f PBMC generating 5T4 peptide specific responses amongst all donors were re-stimulated with the 17 immunogenic 5T4 peptides in the presence of MHC blocking antibodies (L243, 1A3 and W6/32 which block HLA-DR, HLA-DQ and HLA-A/B/C respectively). Significant differences are indicated (***P < 0.0001). Results are expressed as the number of IFN-γ⁺ SFC/10⁵ cultured PBMC after subtracting background spots

Fig. 3

Regulatory T cells do not suppress all effector anti-5T4 T cell responses. a Whole PBMC were depleted of CD25^hi T cells, a representative example of the FACS plots confirming adequate depletion is shown. The proportion of CD4⁺ T cells expressing CD25^hi (b) or Foxp3 (c) in all donors is shown before and after depletion. PBMC and Treg-depleted PBMC from CRC patients were stimulated with each of the five immunogenic peptides (d) and 12 candidate peptides (e). The IFN-γ spot-forming cells/10⁵ cultured PBMC, after subtraction of background spots, was measured before and after the depletion of CD25^hi T cells. f PBMC were cultured with 5T4 peptide 2, with or without the ‘Treg peptide pool’ before restimulation with peptide 2 alone on IFN-γ ELISpot. Assay was carried out in three healthy donors (twelve PBMC lines per condition)